Kidney allografts are naturally tolerogenic in mice. Mice develop tolerance of kidney allografts across full MHC incompatibilities without immunosuppression. This tolerance is initially dependent Foxp3+ cells which are concentrated in the graft in distinctive Treg-rich organized lymphoid structures (TOLS). Recipients of allograft kidneys develop systemic tolerance to skin and heart allografts. This project will identify the immunobiologic features for induction of heart allograft tolerance by kidney allografts in a sequential series of experiments that will determine the timing of systemic tolerance, the dependence on the continued presence of the allograft kidney and thymus, whether the kidney promotes regulatory and/or deletional tolerance and the hypothesized role of kidney dendritic cells, tubular cells and kidney related mediators. We will test for dependence on Foxp3+ cells using B6.Foxp3DTR recipients. We will test whether generation of Foxp3+ cells is related to donor DCs in the kidney and whether adoptively transferred plasmacytoid DCs (pDCs) from the kidney are sufficient to induce acceptance of heart allografts. Renal tubular epithelial cells (RTEC) will be tested in vitro and in vivo for their ability to promote differentiation into Treg and whether selective RTEC injury affects tolerance induction. Infiltrates in cardiac allografts with or without co-transplanted kidneys will be compared for presence of TOLS, pDCs and activated Treg. Finally the mechanisms responsible for the resistance of kidney allografts to sensitized T cells will sought, whether the kidney promotes conversion of T cells to Treg or inactivates or deletes T cells, or is resistant to their action. The kinetics and distribution of Treg and sensitized T cells in the grafts, lymphoid organs and blood will be assessed using in vivo bioluminescence imaging. The fate of the transferred cells in the kidney allograft and their interactions with DCs will be assessed. These studies are expected to reveal mechanisms by which the kidney promotes tolerance of heart grafts, thereby helping to clarify and interpret the observations made in Projects 1 and the EPO experiments in Project 3 and guide the development of clinically applicable treatment protocols.